Traditional live anti-viral vaccines typically require no immunological adjuvants. Similarly, killed virus vaccines are generally much more immunogenic than attenuated pathogen or subunit protein vaccines and, like live anti-viral vaccines, can be effective with no adjuvant or with adjuvants that have limited ability to stimulate immune responses. Recently developed attenuated pathogen or subunit protein vaccines, while offering significant advantages over the traditional vaccines in terms of safety and cost of production, generally have limited immunogenicity compared to whole viruses. As a result, these vaccines typically require adjuvants with significant immunostimulatory capability to reach their full potential in preventing disease.
A number of immunological adjuvants are known in the art, several of which will be briefly mentioned here.
Aluminum salts (alum) have been useful for some vaccines including hepatitis B, diphtheria, polio, rabies and influenza, but may not be useful for others, especially if stimulation of cell-mediated immunity is required for protection. Reports indicate that alum failed to improve the effectiveness of whooping cough and typhoid vaccines and provided only a slight effect with adenovirus vaccines. Problems with alum include induction of granulomas at the injection site and lot-to-lot variation of alum preparations.
Complete Freund's adjuvant (CFA) is a powerful immunostimulatory agent that has been successfully used with many antigens on an experimental basis. CFA includes three components: a mineral oil, an emulsifying agent, and killed mycobacteria, such as Mycobacterium tuberculosis. Aqueous antigen solutions are mixed with these components to create a water-in-oil emulsion. Although effective as an adjuvant, CFA causes severe side effects primarily due to the presence of the mycobacterial component, including pain, abscess formation and fever. CFA, therefore, is not used in human and veterinary vaccines.
Incomplete Freund's adjuvant (IFA) is similar to CFA but does not include the bacterial component. IFA, while not approved for use in the United States, has been used elsewhere in human vaccines for influenza and polio and in veterinary vaccines for rabies, canine distemper and foot-and-mouth disease. However, evidence indicates that both the oil and emulsifier used in IFA can cause tumors in mice.
Muramyl dipeptide (MDP) has been found to be the minimal unit of the mycobacterial cell wall complex that generates the adjuvant activity observed with CFA. See, e.g., Ellouz et al., Biochem. Biophys. Res. Commun. (1974) 59:1317. Several synthetic analogs of MDP have been generated that exhibit a wide range of adjuvant potency and side effects. For a review of these analogs, see, Chedid et al., Prog. Allergy (1978) 25:63. Representative analogs of MDP include threonyl derivatives of MDP (Byars et al., Vaccine (1987) 5:223), n-butyl derivatives of MDP (Chedid et al., Infect. Immun. 35:417), and a lipophilic derivative of a muramyl tripeptide (Gisler et al., in Immunomodulations of Microbial Products and Related Synthetic Compounds (1981) Y. Yamamura and S. Kotani, eds., Excerpta Medica, Amsterdam, p. 167). One lipophilic derivative of MDP is N-acetylmuramyl-L-alanyl-D-isogluatrninyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE). This muramyl tripeptide includes phospholipid tails that allow association of the hydrophobic portion of the molecule with a lipid environment while the muramyl peptide portion associates with the aqueous environment. Thus, the MTP-PE itself is able to act as an emulsifying agent to generate stable oil-in-water emulsions. MTP-PE has been used in an emulsion of 4% squalene with 0.008% TWEEN 80®, termed MTP-PE-LO (low oil), to deliver the herpes simplex virus gD antigen with effective results (Sanchez-Pescador et al., J. Immunol. (1988) 141:1720-1727), albeit poor physical stability.
Recently, MF59, a safe, highly immunogenic, submicron oil-in-water emulsion which contains 4-5% w/v squalene, 0.5% w/v Tween® 80, 0.5% Span®85, and optionally, varying amounts of MTP-PE, has been developed for use in vaccine compositions. See, e.g., Ott et al., “MF59—Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp. 277-296.
QS21, a saponin extracted from the bark of the South American soap bark tree Quillaja saponaria Molina, is another adjuvant that has been shown to have significant immunological activity (Kensil, et al., 1991; Wu, et al., 1992; White, et al., 1991). See, e.g., White, A. C., Cloutier, P. and Coughlin, R. T. A purified saponin acts as an adjuvant for a T-independent antigen. Adv. Exp. Med. Biol. 303:207-210, 1991; Wu, J. Y., Gardner, B. H., Murphy, C. I., Seals, J. R., Kensil, C. R., Recchia, J., Beltz, G. A., Newman, G. W. and Newman, M. J. Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-I vaccine. J. Immunol. 148:1519-1525, 1992; Kensil, C. R., Patel, U., Lennick, M. and Marciani, D. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. J. Immunol. 146:431-437, 1991.
Immunostimulating complexes (ISCOMs) containing a saponin, a sterol and, optionally, a phospholipid are also known. For example, U.S. Pat. No. 4,900,549 teaches a process for preparing immunogenic complexes containing an amphiphatic antigenic protein or peptide, a sterol, and a glycoside comprising hydrophobic and hydrophilic regions. Optionally, the complexes also contain a phospholipid, preferably phosphatidylethanolamine. The preferred sterol is cholesterol, and preferred glycosides are saponins, especially Quil A (a Quillaja saponaria Molina saponin extract). Methods for producing ISCOMs are known in the art and described in e.g., U.S. Pat. No. 5,118,671, U.S. Pat. No. 4,900,549, International Publication No. WO 90/103184 and Bomford et al. Vaccine (1992) 10:572-577. Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMs as the delivery vehicle for antigens (Mowat and Donachie, Immunology Today 12:383-385, 1991). Doses of antigen as low as 1 ug encapsulated in ISCOMS have been found to produce class I mediated CTL responses, where either purified intact HIV-1-IIB gp160 envelope glycoprotein or influenza hemagglutinin is the antigen (Takahashi et al., Nature 344:873-875, 1990).